The present invention relates to methods and compositions for regulating the activity of imidazoline receptors. In particular, the invention relates to pharmaceutical compositions comprising imidazoleacetic acid-ribotide (IAA-RP) and imidazoleacetic acid-riboside (IAA-R). The invention is based on the discovery that IAA-RP and to a lesser extent IAA-R bind with a high affinity to imidazoline receptors. Antibodies to IAA-RP and IAA-R are additionally provided, as well as screening methods for identification of compounds that either promote or antagonize the activity of IAA-RP and IAA-R. The invention further relates to diagnostic and prognostic methods for detection of abnormalities in levels or activity of IAA-RP and IAA-R. The invention encompasses treatment of disorders related to the imidazoline system, including hypertension, glaucoma, psychiatric (e.g., depression), neurological (e.g., motor disorders, neurodegenerative disorders), diabetes and disorders related to platelet aggregation.
Imidazoline receptors are now generally recognized as a unique set of non-adrenergic high affinity binding sites for a number of agents that to date also bind to xcex12-adrenergic receptors (Eglen, R. M. et al., 1998, Trends in Pharmacol. Sci. 19: 381-390; Regunathan, S. and Reis, D. J., 1996, Ann. Rev. Pharmacol. Toxicol. 36:511-44). Although membrane-bound imidazoline receptors have not yet been cloned, evidence including differences in selectivity and binding affinity of ligands, the structure of binding proteins and cellular distribution indicate that they are different from xcex12-adrenergic receptors. The nonadrenergic imidazoline receptors are important in mediating the hypotensive actions of clinically important imidazoline drugs such as clonidine, rilmenidine and moxonidine.
For example, unique imidazoline receptors, are present in pancreatic islet and beta-cells (Morgan, N. G., et al., 1995, Ann. N.Y. Acad. Sci. 763: 361-373). Activation of these receptors by imidazolines causes release of insulin. Much of this activity is due to imidazoline-induced closure of K+ channels such as the K+ ATP-sensitive channels which permits intracellular levels of K+ to accumulate, causing cell depolarization and eventual exocytosis of hormone or transmitter into plasma or extracellular fluid. It is noteworthy that channels such as the K+ ATP-sensitive channels exist throughout the body, and are particularly abundant in brain. These pancreatic imidazoline receptors have recently been designated as 13 receptor subtypes (Eglen, R. M. et al., 1998, TIPS 19: 381-390). Chan et al. (1997, Brit. J. Pharmacol. 120: 926-932), showed that imidazolines and preparations of CDS (clonidine-displacing substance, see below) from bovine brain caused release of insulin and stimulated K+ ATP channels.
One or more endogenous ligands selectively bind to the imidazoline receptors although attempts to identify this endogenous ligand(s) has failed. A possible ligand, referred to as xe2x80x9cclonidine displacing substancexe2x80x9d (CDS), has been discovered as an entity isolated from mammalian brain and the periphery that is capable of displacing radio-labeled clonidine and its radio-labeled congeners from membranes (Atlas, D. et al., 1987, J. Cardiovascular Pharmacology 10(Suppl. 12): S122-S127; Atlas, D. 1991, Biochemical Pharmacology 41: 1541-1549; Atlas, D., 1995, Annals of the New York Academy of Sciences 763:314-324). Antibodies have been prepared against the drug clonidine, which presumably interact with CDS. Such antibodies are found to be immunoreactive in tissues throughout the body and also show a heterogeneous regional distribution within the brain.
A recent study proposed that agmatine, a known compound isolated from bovine brain, is CDS (Li, G. et al., 1994, Science 263:966-968; Regunathan, S. and Reis, D. J., 1996, Ann. Rev. Pharmacol. Toxicol. 36: 511-544; but see Eglen, R. M. et al., 1998, TIPS 19: 381-390). Agmatine was further suggested to be an endogenous neurotransmitter because it was found within an extract of CDS activity from whole brain and because it appeared to bind to a class of imidazoline receptors. However, comparisons of the biological activities of agmatine, e.g., effects on blood pressure versus effects of endogenous clonidine-displacing substance at imidazoline and xcex12-adrenergic receptors produced in virtually all laboratories indicated that agmatine differed from xe2x80x9cclassical CDS.xe2x80x9d For example, agmatine displaces labeled clonidine from a subset of its nonadrenergic binding sites identified as imidazoline 2A and 2B sites. However, because those I2A and I2B sites are now known to be enzymes, i.e. portions of monoamine oxidase A and B, the search for the identity of CDS that acts at membrane-bound imidazoline receptors has continued (Eglen, R. M. et al., 1998, TIPS 19: 381-390).
Several laboratories have harvested CDS and most preparations show similar physiochemical properties. There is widespread consensus that CDS is present in small amounts in the brain, cerebrospinal fluid and periphery (including plasma) of mammals. It is soluble in water and methanol, but generally insoluble in organic solvents. Size exclusion chromatography indicated that it is a small molecule (xe2x89xa61000 Da). CDS is resistant to several proteases, including trypsin and chymotrypsin, and is devoid of amino acids; thus it is not a peptide. CDS appears to have no free amino groups as activity is retained following reaction with fluorescamine and ninhydrin. CDS is stable in both weak acids (pH 2) and weak bases (pH 10.5), is thermostable (at 110xc2x0 C.) and retains activity following multiple freeze-thaw and lyophilization cycles. Because CDS can be retained on both anion and cation exchange resins and because its migration patterns shifted markedly with changes in ambient pH on gel electrophoresis, it is very likely that CDS is amphoteric, possibly a zwitterion. In addition, CDS shows maximal UV absorbance between 206-220 nm.
The present invention relates to methods and compositions for regulating the activity of imidazoline receptors. Specifically, the invention relates to compositions comprising imidazoleacetic acid-ribotide (IAA-RP) which binds with high affinity to at least 2 subsets of imidazoline receptors, imidazoleacetic acid-riboside (IAA-R) which binds with a slightly lower affinity, and to its related congeners. As demonstrated herein, IAA-RP binds to imidazoline receptors and in one case stimulates well defined receptor-mediated signal transduction events such as release of arachidonic acid. Further, the release of arachidonic acid, an imidazoline I1-receptor mediated event, is inhibited in the presence of the imidazoline I1 receptor antagonist efaroxan. The discovery that IAA-RP and IAA-R bind to imidazoline receptors provides new targets for therapeutic methods aimed at amelioration of imidazoline system related disorders.
The present invention includes pharmaceutical compositions comprising IAA-RP, IAA-R, derivatives and analogs thereof, which can be utilized to regulate the activity of imidazoline and imidazoline-like receptors. Such compositions can be utilized to treat disorders related to the imidazoline system such as hypertension, glaucoma, psychiatric (e.g. depression), neurological (e.g. motor disorders, neurodegenerative disorders), diabetes and disorders involving platelet aggregation.
The invention further provides for antibodies to IAA-RP and IAA-R. Such antibodies can be utilized to ameliorate symptoms associated with imidazoline system-related disorders. For example, in the case of an anti-IAA-RP antibody, such an antibody would specifically bind to IAA-RP and possibly disrupt the ability of IAA-RP to bind to imidazoline receptors thereby preventing receptor mediated signal transduction events. Additionally, anti-IAA-RP and anti-IAA-R antibodies can be used as diagnostic and prognostic indicators of imidazoline system related disorders. For example, diagnostic methods can be utilized to detect abnormalities in the levels or tissue distribution of IAA-RP and/or IAA-R relative to normal levels. The antibodies of the invention can also be used in screening methods for detection of a predisposition to imidazoline system based disorders in an individual.
The invention further relates to methods for identification of compounds which promote or antagonize signal transduction events stimulated by the binding of IAA-RP or IAA-R to imidazoline receptors. Such compounds can act as therapeutic agents in the amelioration of a wide range of imidazoline system based disorders. The invention further relates to methods for identification of compounds that regulate the synthesis, or degradation of IAA-RP or IAA-R.
Finally, the invention relates to treatment of imidazoline based disorders, such as for example, hypertension, glaucoma, psychiatric (e.g. depression), neurological (e.g. motor disorders, neurodegenerative disorders), diabetes and disorders involving platelet aggregation by administering compositions comprising IAA-RP, IAA-R, or compounds that promote or antagonize IAA-RP or IAA-R activity.